1. Field of the Invention
The invention relates to a new monoclonal antibody directed against the transferrin receptor (TfR), and to its use for the immunotherapy of hyperproliferative diseases, such as cancer.
2. Description of the Background
The transferrin receptor (CD71) is a disulfide-linked homodimeric transmembrane glycoprotein consisting of two 760-amino acid monomers of approximately 90 kDa each. TfR plays a crucial role in the regulation of iron uptake and cell growth (GILL et al., N Engl J. Med., 332, 1744-1748, 1995—HERMINE et al., N Engl J. Med., 332, 1749-1751, 1995). When diferric transferrin binds to its cell surface receptor, it is internalized via clathrin-coated pits to acidic vesicles where the iron-transferrin complex is dissociated. After release, the receptor and apo-transferrin recycle back to the cell surface.
TfR is constitutively expressed in cells of tissues that are constantly renewed, such as precursors of blood cells in the bone marrow, hepatocytes in the liver, keratinocytes in the epidermis and enterocytes in crypts of intestinal epithelium. Several studies have shown that TfR is expressed more abundantly in malignant tissues than in their healthy counterparts (GATTER et al., J Clin Pathol., 36, 539-545, 1983—FAULK et al, Lancet., 2, 390-392, 1980—SHINDELMAN et al, Int J Cancer, 27, 329-334, 1981).′ Several authors have reported therapeutic approaches based on this idea using anti-TfR antibodies or transferrin itself conjugated to drugs to kill malignant cells.
It has also been proposed to use anti-TfR antibodies to block the interaction between transferrin and TfR, and consequently iron uptake, leading to iron deprivation and negative regulation of cell growth. However, although many publications describe the preparation of anti-TfR antibodies, there are very few reports of anti-TfR monoclonal antibodies (mAbs) having an antiproliferative activity.
TROWBRIDGE and LOPEZ (Proc. Natl Acad Sci USA, 79, 1175-1179, 1982) report the properties of a monoclonal antibody, designated 42/6 and typed as IgA (k), that blocks the binding of transferrin to its receptor and is able to inhibit in vitro the growth of an human T leukemic cell line, by blocking the cells in S phase of the cell cycle. The 42/6 antibody and the hybridoma producing it (ATCC HB-8094) are disclosed in U.S. Pat. No. 4,434,156.
TAETLE et al. (Cancer Res., 46, 1759-1763, 1986) have compared the 42/6 antibody with another anti-transferrin receptor monoclonal antibody (B3/25), which belongs to the IgG class, does not block the binding of transferrin to its receptor, and does not inhibit lymphocyte growth. They observed that transferrin did not inhibit 42/6 binding, suggesting that the inhibition of transferrin binding by antibody 42/6 inhibited results from a non-competitive, possibly steric, mechanism.
The 42/6 antibody has been tested in a Phase Ia trial, and it was observed that it was generally well tolerated, and that antibody concentrations that were capable of inhibiting malignant blood cell growth were obtained without toxicity. (BROOKS et al., Clin Cancer Res., 1, 1259-1265, 1995).
It was initially suggested that the antiproliferative properties of the 42/6 antibody were the result of its ability to block transferrin binding, thus depriving the cells of iron, and that other antibodies having the same ability would have the same antiproliferative properties. However, further studies have shown that the situation was more complicated.
WHITE et al. (Cancer Res., 50, 6295-301, 1990) and U.S. Pat. No. 5,667,781 disclose the effects of 42/6, B3/25, and 32 other monoclonal antibodies directed against the external domain of TfR (all of the IgG class), on the binding of transferrin to its receptor and on the growth of leukemic T-cells in tissue culture. No antibody other than 42/6 was found to be able to inhibit transferrin binding. Most of the IgG antibodies had no or little inhibitory effect on the growth of leukemic T-cells. Only one of them designated D65.30 and belonging to the IgG1 isotype, showed growth-inhibitory properties similar to those of 42/6, although it did not inhibit the binding of transferrin to TfR. Combinations of two or more of these anti-TfR monoclonal antibodies were also tested. Some of them did not differ in their growth-inhibitory effects from the individual antibodies, other were antagonistic, and some of them showed a synergistic growth-inhibitory effect. Some of these combinations showed a cytotoxic action, while the individual antibodies had a cytostatic action, or were inactive. These combinations of monoclonal antibodies with high antiproliferative properties did not block transferrin binding to TfR. Prolonged incubation, (48 h) of cells with the antiproliferative combinations B3/25 and 42/6, or D65.30 and A27.15, or with the individual antibodies B3/25, 42/6, or D65.30 induced a down-regulation of surface TfR expression resulting in a reduction of Tf binding at 4° C. and 37° C. However only 42/6 alone, and the combinations B3/25 and 42/6, or D65.30 and A27.15 were able to reduce TfR internalization.
LESLEY et al. (Mol Cell Biol. 5, 1814-21, 1985) have studied the effects of anti-murine transferrin receptor monoclonal antibodies belonging to either the IgG or the IgM class, on binding of transferrin and on murine lymphoma cell growth in vitro. They observed that the IgM inhibited cell growth but that the IgG did not, although they were able to induce a down-regulation and a degradation of the TfR. However, IgG cross-linked by an antiimmunoglobulin antibody were able to inhibit cell growth. In a subsequent work, the same team (LESLEY et al., Exp Cell Res., 182, 215-33, 1989) have studied the effects of IgG and IgM monoclonal anti-TfR antibodies and of their mono- and divalent fragments on murine lymphoma cell growth and TfR expression. They report that these effects depend on the degree of crosslinking of transferrin receptors by the antibody, which is a consequence of the antibody valence. Monovalent antibody fragments had no significant effects; divalent antibody fragments induced a down-regulation of cell-surface receptor expression without impairing its internalization and recycling and without impairing cell growth; multivalent IgM induced the accumulation of antibody-complexed receptor on the cell surface, blocking its internalization and resulting in a strong inhibition of cell growth.
It appears from the prior art cited above that the anti-proliferative properties of anti-TfR antibodies strongly vary from an antibody to another and that they cannot be predicted on the basis of their ability to block or not transferrin binding to its receptor.
In a previous publication (MOURA et al., J Exp Med, 194, 417-425, 2001), the Inventors have reported that a monoclonal IgG (IgG2kappa), designated A24, produced by one of the hybridomas obtained from a mouse hyperimmunized with IgA-binding proteins, was in fact directed against the human TfR.